Quantitative Cu-Homeostasis in Live Mammalian Cells at the Single-Molecule Level

项目概要/摘要 了解金属蛋白的反应机制是阐明铜生物学功能的关键 并确定由异常金属体内平衡引起的疾病的原因。细胞铜吸收和 分泌需要相关的金属蛋白以时空协调的方式进行调节， 细胞铜水平。然而，在铜领域，很少有人知道金属蛋白是如何单独调控 也不能在它们的原生环境中系统地相互合作，即，在细胞中。我们的研究目标是 了解活的哺乳动物细胞中铜摄取和分泌金属蛋白的反应机制， 特别关注金属蛋白如何调节它们的行为，如空间分布，寡聚状态， 蛋白质间和结构域间相互作用，以时空限定的方式实现适当的Cu平衡。先前 PI的成就包括发现MerR家族金属调节剂在调节 通过检查蛋白质-DNA相互作用的外排泵的转录和Cu响应动态组装 和蛋白质扩散行为在活细菌中使用单分子超分辨率显微镜。利用 这些技术的力量，结合最近开发的活细胞单分子荧光， 共振能量转移分析，我们将阐明金属蛋白在摄取的响应机制 和分泌途径。使用CTR 1和ATOX 1-ATP 7 A/B作为 摄取和分泌金属蛋白，所提出的实验将（1）量化铜依赖性低聚状态 分布和鉴定CTR 1的Cu响应部分;（2）定义ATOX 1与 ATP 7A和ATP 7 B以及ATP 7 B突变如何影响细胞Cu防御中的Cu稳态 多能干细胞衍生的肝细胞。除了单分子超分辨的主要方法外， 荧光成像技术，互补的批量光谱和生化测量将是 比较了该研究计划通过与铜稳态专家的合作得到进一步加强， 干细胞领域这项研究意义重大，因为它将为金属蛋白质提供机理上的见解- 介导的Cu吸收和分泌过程以及同步加速器X射线的补充信息 细胞内Cu再分布的荧光研究。人诱导性多能干细胞的比较 细胞（hiPSC）衍生的健康和患病肝细胞将告知疾病突变如何破坏细胞Cu 平衡，提供知识基础，以制定威尔逊病的治疗策略。这项研究是 创新，因为它通过将重点转移到确定应对措施， 使用先进的方法，包括单分子超分辨率的金属蛋白的机制 显微镜和hiPSC衍生的肝细胞。活细胞成像方法也绕过了一般的 研究膜复合物的挑战，其体外重建在技术上是苛刻的。hiPSC- 来源的病变肝细胞为研究Wilson病的发病机制提供了理想的平台。

PROJECT SUMMARY/ABSTRACT Understanding responsive mechanisms of metalloproteins is key to elucidate biological functions of copper (Cu) and to identify the causes of diseases resulting from abnormal metal homeostasis. The cellular Cu uptake and secretion require relevant metalloproteins to adjust in a spatiotemporally coordinated manner to assure proper cellular Cu level. However, in the Cu field, little is known about how metalloproteins are individually regulated nor systematically cooperate with each other in their native environment, i.e., in cells. Our research goal is to understand the responsive mechanisms of Cu-uptake and secretory metalloproteins in live mammalian cells, with specific focuses on how metalloproteins adjust their behaviors such as spatial distributions, oligomeric states, inter-protein and inter-domain interactions for proper Cu balance in a spatiotemporally defined manner. Previous achievements of the PI include discoveries of novel mechanisms of MerR-family metalloregulators in regulating transcription and Cu-responsive dynamic assembly of efflux pumps by examining the protein-DNA interaction and protein diffusive behaviors in live bacteria using single-molecule super-resolution microscopy. Leveraging the power of these technologies, in combined with the recently developed live-cell single-molecule fluorescence- resonance-energy-transfer assay, we will elucidate the responsive mechanisms of metalloproteins in the uptake and secretory pathways in live mammalian cells. Using CTR1 and ATOX1-ATP7A/B as the initial examples of uptake and secretory metalloproteins, the proposed experiments will (1) quantify Cu-dependent oligomeric state distribution and identify the Cu-responsive moiety of CTR1; (2) define the preferential interaction of ATOX1 to ATP7A and ATP7B and how mutations in ATP7B affect Cu homeostasis in cellular Cu defending using induced pluripotent stem cells derived hepatocytes. In addition to primary approaches of single-molecule super-resolution fluorescence imaging techniques, complementary bulk spectroscopic and biochemical measurements will be compared. The research program is further enhanced by collaborations with the experts in Cu homeostasis and stem cell fields. The research is significant because it will provide mechanistic insights into metalloprotein- mediated Cu-uptake and secretion processes as well as complementary information for synchrotron X-ray fluorescence studies on intracellular Cu-redistribution. The comparison between human induced pluripotent stem cell (hiPSC)-derived healthy and diseased hepatocytes will inform how disease mutations disrupt cellular Cu balance, providing the knowledge base to devise therapeutic strategies for Wilson's diseases. The research is innovative because it represents a substantive departure from the status quo by shifting focus to define response mechanisms of metalloproteins using advanced approaches including single-molecule super-resolution microscopy and hiPSC-derived hepatocytes. The live-cell imaging approach also circumvents the general challenge in studying membrane complexes, whose in vitro reconstitution is technically demanding. The hiPSC- derived diseased hepatocytes provide an ideal platform to study the pathogenesis of Wilson's disease.